RESULTS: Spheroids generated from nasopharyngeal carcinoma cell line HK-1 continuously grew and invaded into collagen matrix in a 24-well plate. Similar observations were noticed with spheroids embedded in the 3D chip. Large spheroids in both 24-well plate and the 3D chip disintegrated and invaded into the collagen matrix. Preliminary drug sensitivity assays showed that the growth and invasion of spheroids were inhibited when spheroids were treated with combination of cisplatin and paynantheine at high concentrations, in a 24-well plate. Comparable findings were obtained when spheroids were treated with the same drug combination in the 3D chip. Moving forward, spheroid assays could be performed in the 3D chip in a more high-throughput manner with minimal time and cost.
METHODS: We compared the ability of Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) and Alpha Minimum Essential Medium (α-MEM) with Glutamax (GL) (α-MEM/GL) to expand hUCM cells. For this purpose, hUCM cells were cultured in plates containing different culture media supplemented with 10% fetal bovine serum (FBS). Culture dishes were left undisturbed for 10-14 days to allow propagation of the newly formed hUCM cells. The expansion properties, CD marker expression, differentiation potential, population doubling time (PDT) and cell activity were compared between the two groups.
RESULTS: The hUCM cells harvested from each group were positive for MSC markers, including CD44, CD90 and CD105, while they were negative for the hematopoietic cell surface marker CD34. Differentiation into adipogenic and osteogenic lineages was confirmed for both treatments. Cell activity was higher in the α-MEM/GL group than the DMEM/F12 group. PDT was calculated to be 60 h for the DMEM/F12 group, while for the α-MEM/GL group it was 47 h.
CONCLUSIONS: Our data reveal that α-MEM/GL with 10% FBS supports hUCM cell growth more strongly than DMEM/F12 with 10% FBS.
METHODS AND RESULTS: In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR 219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25 ± 2 °C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants.
CONCLUSION: This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice.
RESULTS: The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.
CONCLUSIONS: Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.